Every year, nearly 100,000 patients need coronary or peripheral revascularization procedures, but have no suitable vein or artery. The tissue engineering of autologous vessels for these patients could have an enormous impact on therapy for cardiovascular disease. We have developed techniques to culture tissue engineered vessels from differentiated vascular cells. Engineered arteries are cultured on degradable polymer scaffolds in pulsatile perfusion systems. Vessels that are produced are strong enough for initial implantation. Completely autologous animals grafts, as well as human engineered vessels, have cultured using these methods. However, implanted autologous vessels dilate significantly over time in vivo, due to limitations in ultimate mechanical strength. In addition, engineered graft survival in animal models is limited by thrombosis. Thus, although significant advances have been made in vascular tissue engineering, mechanical strength and thrombogenicity limit further progress in the field. Collagen determines the ultimate mechanical properties of blood vessels. Engineered blood vessels have less collagen than native vessels, and this collagen has lower load-bearing properties than native. We postulate that the "defective" collagen in engineered vessels is caused by inadequate cross-linking, and by excessive cleavage by proteases. This proposal will test this overall hypothesis. Specifically, we will study the effects on collagen cross-linking of copper supplementation to increase lysyl oxidase activity. In addition, we will study the effects of metalloproteinase inhibition on collagen accumulation and vessel strength, and we will assess the effects of retinoids on cross- linking activity, metalloproteinases, and cellular quiescence in engineered vessels. These experiments will thus work synergistically to improve tissue engineered collagen, and hence improve vessel mechanical strength. The other important limitation for engineered vessels is early thrombosis. Cultured endothelial cells decrease expression of thrombomodulin, which is an important anti-coagulant in the native arterial system. We will examine the functional significance of thrombomodulin loss in engineered vessels. With the aim of improving the patency of these vascular grafts, we will also quantify the effects of thrombomodulin over-expression on vessel thrombogenicity. Thus, by attacking the two important impediments in vascular tissue engineering, the experiments in this proposal will significantly advance the field.